1. Field of the Invention
This invention relates to methods for extraction and concentration of water and oil soluble compounds from a biological sample using liquid carboxylic acids.
2. Description of the Related Art
Salmonella serotype enteritidis (SE) emerged as a major cause of human salmonellosis in the United States throughout the 1980s and 1990s. Of the 360 SE outbreaks with a confirmed source, 279 (82%)) were associated with raw or undercooked shell eggs between 1985 and 1998 (CDC, 2000). Grade-A shell eggs have been attributed to the human salmonellosis problem due to the ability of SE to infect ovarian tissues and to be deposited into the developing egg. It is difficult to detect flocks or individual birds that may be infected with SE and lay contaminated eggs since laying hens seldom show any clinical symptom of SE infection. Studies indicate that the percentage of infected eggs is extremely low and sporadic with most contaminated eggs having small number of SE (Humphrey, 1994). Culturing both environmental samples and bird tissues does not prove the birds are laying SE infected eggs. The only method of directly proving that a flock is laying SE-contaminated eggs is by culturing the organism from eggs.
Conventional methods for detection of Salmonella from eggs take 5-7 days and are labor-intensive, involving isolation of the organism using pre-enrichment as well as selective enrichment procedures and serological confirmation tests. Detection of small numbers of SE in inoculated pools of egg contents was successful using direct plating of incubated egg pools onto agar plates although the more enrichments steps applied, the better the sensitivity achieved (Gast, Poultry Science, 72, 1611-1614, 1993). Supplementing pools of egg contents with iron in the form of ferrous sulphate and concentrated enrichment broth has been suggested to improve detection of SE from raw eggs without using enrichment broth (Gast and Beard, Poultry Sci., Volume 70, 1273-1276 1991; Cudjoe et al., Int. J. Food Microbiology, Volume 23, 149-158, 1994; Gast and Holt, Jour. Food Prot., Volume 61, 107-109, 1998). More rapid methodologies have been developed using ELISA and PCR techniques (Holt et al., J. Food Prot., Volume 58, 967-972, 1995; Woodward and Kirwan, Vet. Rec., Volume 138, 411-413, 1996). In contrast to conventional methods, these tests can detect SE in two days. However, they are not free of drawbacks. The tests involve time-consuming enrichment incubations, exhibit varying degrees of cross-reactions, and both systems have been known to produce false positive reactions. The sensitivity of rapid detection methods for SE in eggs was substantially decreased due to interference of egg contents (Brigmon et al., Poultry Science, Volume 74, 1232-1236, 1995). Cudjoe et al. (1994, supra) found that the more viscous, undiluted mixtures of eggs showed the highest particle loss compared with diluted samples when immunomagnetic beads were applied to recover SE from raw eggs.
U.S. Pat. No. 5,367,054 (Young-Zoon, Nov. 22, 1994) discloses methods for isolating and purifying immunoglobulins from eggs which includes a first step of extracting a diluted, homogenized egg yolk with a composition containing one or more medium-chain fatty acids, i.e. any fatty acid having 6-12 carbon atoms, such as caprylic acid, caproic acid, capric acid, and lauric acid while homogenizing the mixture for a second time.
U.S. Pat. No. 5,932,250 (Stolle et al., Aug. 3, 1999) discloses extraction of egg yolk IgY protein fraction using caprylic acid where egg yolk is diluted 7.5 fold with deionized water and then diluted 1:1 with 0.06 M acetate buffer, pH 5. One percent caprylic acid is blended in and the mixture allowed to stand for 2 hours for separation of the aqueous (bottom) layer and the oil solubles (top layer); with immunoglobulin in the aqueous layer. The bottom layer is neutralized and diafiltered and concentrated.
McLaren et al. (Journal of Immunological Methods, Volume 177, 175-184, 1994) disclose extraction of a diluted pool of egg yolk with a final caprylic acid concentration of 6% (v/v). After the addition of caprylic acid, the mixture was stirred for two hours at room temperature followed by centrifugation to obtain a supernatant containing immunoglobulin.
While various methods have been disclosed for obtaining proteins, such as immunoglobulins, from biological samples, such as egg yolk, there remains a need in the art for methods for rapid extraction of proteins, such as immunoglobulins and antigens; and infectious agents such as bacteria, from biological samples. The present invention provides a method for extracting such proteins and/or infectious agents from biological samples which is fast, provides a concentrated sample, provides increased sensitivity when used in diagnostic testing, and is different from prior art methods and solves some of the problems associated with prior art extraction methods.